Selection of internalizing human antibodies targeting pancreatic tumor cells in s

描述（由申请人提供）：本提案的目的是鉴定内化的人单链抗体（scFvs），该抗体原位靶向胰腺肿瘤细胞，重点是(1)早期肿瘤（0/I期），内化scFvs可用于无创成像以进行早期检测，以及(2)晚期肿瘤（III/IV期），内化scFvs可用于开发基于细胞内递送策略的靶向治疗。目前，很少有针对胰腺肿瘤的人单克隆抗体，而专门检测早期胰腺肿瘤以实现无症状肿瘤的无创成像的人单克隆抗体就更少了，这一领域对治疗方案和结果有着巨大的影响。因此，我们提出的研究将解决这一领域的一个关键需求。我们假设像其他肿瘤一样，胰腺肿瘤具有独特的细胞表面表位谱，将肿瘤与非肿瘤组织区分开来。其中一些表位可能在肿瘤进展早期出现，从而可以早期发现无症状的肿瘤。我们进一步假设，这些肿瘤细胞表面表位的一个子集是内化的，因此可以用来向胰腺肿瘤细胞内传递有效载荷。由于肿瘤细胞表面表位空间是由复杂的抗原决定因子组成的，包括翻译后修饰，我们采取了基于抗体的方法来研究肿瘤细胞表面。我们之前已经开发并利用了包含5亿个成员的幼稚噬菌体抗体文库来鉴定针对肿瘤相关内化表位的人单链抗体（scFvs），促进了基于细胞内递送策略的靶向治疗的进一步发展。认识到肿瘤细胞系通常表达与原位肿瘤细胞不同的细胞表面抗原谱，我们最近开发了一种新方法，利用激光捕获微解剖（LCM）在原位肿瘤细胞上选择噬菌体抗体文库，从而产生针对临床代表的肿瘤抗原的抗体，而不是与体外培养的细胞系相关的可能伪影。通过使用这些技术，我们能够鉴定出针对临床标本中原位表达的肿瘤抗原的人单克隆抗体，并具有新颖的、治疗上有用的功能，如靶向细胞内有效载荷递送。通过LCM精确获取疾病细胞，使不同阶段的肿瘤细胞成为噬菌体抗体选择的目标，为鉴定可能用于早期肿瘤检测的阶段相关抗体提供了机会。我们建议将这一策略应用于选择内在化的人scFvs，这些scFvs在不同阶段靶向胰腺肿瘤细胞。这些新型抗体是人类序列，未来可用于开发靶向治疗和基于非侵入性成像的早期肿瘤检测。公共卫生相关性：本项目旨在通过激光捕获显微解剖鉴定靶向胰腺肿瘤细胞的内在化人单链抗体。这些内化的scFvs可用于开发非侵入性成像策略，从而实现敏感和准确的早期肿瘤检测，以及基于细胞内递送策略的靶向治疗。

DESCRIPTION (provided by applicant): The objective of this proposal is to identify internalizing human single chain antibodies (scFvs) that target pancreatic tumor cells in situ, with emphasis on (1) early stage tumors (stages 0/I), for which internalizing scFvs can be used in non-invasive imaging for early detection, and (2) late stage tumors (stages III/IV), for which internalizing scFvs can be used to develop targeted therapy based on intracellular delivery strategies. Currently, there are very few human monoclonal antibodies that target pancreatic tumors, and even fewer that specifically detect early stage pancreatic tumor to allow non-invasive imaging of asymptomatic neoplasia, an area that has a huge impact on treatment options and outcomes. Thus our proposed research would address a critical need of this field. We hypothesize that like other tumors, pancreatic tumors possess unique cell surface epitope profiles that distinguish tumors from non-neoplastic tissues. Some of these epitopes may be present early in tumor progression, allowing early detection of asymptomatic neoplasia. We further hypothesize that a subset of these tumor cell surface epitopes are internalizing, and thus can be utilized to deliver payloads intracellularly to pancreatic tumor cells. Because the tumor cell surface epitope space is composed of complex antigenic determinants including posttranslational modifications, we have taken an antibody-based approach to study the tumor cell surface. We have previously developed and utilized a naive phage antibody library containing 500 million members to identify human single chain antibodies (scFvs) targeting tumor associated internalizing epitopes, facilitating further development of targeted therapy based on intracellular delivery strategies. Recognizing that tumor cell lines often express a different cell surface antigenic profile than that of tumor cells in situ, we have recently developed a novel method that utilizes laser capture microdissection (LCM) to select phage antibody libraries on tumor cells in situ, thereby generating antibodies against clinically represented tumor antigens as opposed to possible artifacts associated with cell lines cultured in vitro. By using these techniques we are able to identify human monoclonal antibodies that target tumor antigens expressed in situ in clinical specimens, and possess novel, therapeutically useful functions such as targeted intracellular payload delivery. The precise procurement of disease cells by LCM allows tumor cells at different stages to be targeted for phage antibody selection, offering opportunities to identify stage-associated antibodies that may be used in early tumor detection. We propose to apply this strategy to select internalizing human scFvs that target pancreatic tumor cells in situ at different stages. These novel antibodies, which are human in sequence, can in the future be used to develop targeted therapeutics and non-invasive imaging-based early tumor detection. PUBLIC HEALTH RELEVANCE: This project aims to identify by laser capture microdissection internalizing human single chain antibodies that target pancreatic tumor cells in situ. These internalizing scFvs can be used to develop non-invasive imaging strategies that allow sensitive and accurate early tumor detection, and targeted therapies based on intracellular delivery strategies.